Unknown Identification
Overview
This lab should give you the background information and techniques you will need to successfully identify your
case study specimens later. The micro lab website, your textbook, the web and assorted books available in lab will
be the reference materials necessary for you to successfully complete the next several weeks of lab work.

Each pair will receive one organism to identify…
 You will be told if you have a staph, a strep or an enteric.
 You will conduct tests appropriate for your organism to determine species identification.

Each pair may have to present information on the specific organism they identified including…
 Test results
 Where it is part of the normal flora
 When and where it becomes a pathogen
 What diseases it causes

Each pair may also be assigned a specific media test to present the following information on
 What organism(s) does it differentiate?
 What do positive results look like?
 What is the biochemical basis of the test?
Lab Procedure
We have included the basic procedure for doing each biochemical test below. You will find more specific
procedures for each biochemical test under the media section of the micro lab website. More complete
information on selective & differential media can be obtained by consulting the Difco manuals in lab. You will
need to look up the individual test for a more detailed description, including the biochemical basis of each test.
Test
Brief Instructions
TSA/BHI
Gram Stain
Motility
Probable Results
Staphs/Enterics on TSA; Streps on BHI
Determine macromorphology
To confirm culture purity
Staphs/Streps (Gram+), Enterics (Gram-)
Stab with a needle straight in and straight out of the Motile organisms have obvious growth
center of the tube half way down. Incubate for 24
away from inoculation area; Non-motile
hours at 37°C. Staphs/Enterics in O2; Streps in CO2.
organisms grow only in inoculation area.
McFarland
Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to a 0.5
Standard
McFarland test standard. Hold your diluted tube and the 0.5 McFarland test standard against
the black-lined McFarland reference card to accurately rate the turbidity.
FTM
Use a sterile transfer pipette to add 1 mL of your
Strict aerobes will grow near the top of
McFarland standard organism in the middle of the
the media; Facultative anaerobes will
tube. Cap tightly; do not jostle. Incubate for 24
grow throughout; Strict anaerobes will
hours at 37°C.
grow near the bottom.
Catalase
Transfer a well-isolated colony to a clean slide & add Staphs/Enterics are catalase positive;
1 drop of 3% H2O2. Do not reverse the order & do
bubble formation should occur. Note: do
not mix. Observe for immediate bubble formation.
not take colony from a blood plate.
Oxidase
Add a few drops of oxidase reagent onto Whatman
Look for appearance of purple color
filter paper. Smear with a loop-full of organisms.
within 10-15 seconds. See probable
Use colonies from low glucose, non-selective media. results table.
Table 1: Brief Description of general tests that every group will do.
1|Unknown Identification
Staphylococcus
The Staph groups will do these specific tests in addition to the general tests from Table 1.
 TGA (Tellurite Glycine Agar)
 Coagulase
 MSA (Mannitol Salt Agar)
 Novobiocin Antibiotic Disk Sensitivity
 Hemolysis (Blood Agar)
Test
Brief Instructions
Probable Results
TGA
Streak for Isolation. Incubate for 24 hours at 37°C.
See probable results table 3 below.
Add a loop-full or 0.5mL of a pure culture to 0.5mL
Coagulase
rabbit plasma. Gently rotate tube to mix, do not
Presence of clot indicates S. aureus
shake. Incubate for 24 hours at 37°C.
MSA
Streak for isolation. Incubate for 24 hours at 37°C.
See probable results table 3 below.
Dilute colonies from a pure culture into sterile
Novobiocin
A zone of growth inhibition ≤16 mm in
saline to a 0.5 McFarland standard. Swab half the
Antibiotic
diameter in a coagulase(-) staph is
surface of a blood agar plate. Place a novobiocin
Disk
indicative of S. saprophyticus. See
disk lightly onto the surface. Incubate for 24 hrs at
Sensitivity
probable results table 3 below.
37°C.
Streak the other half of the blood agar plate to
Beta hemolysis is indicative of S.
Hemolysis
check for hemolysis. Stab into the agar surface at
aureus. See probable results table 3
the last part of your streak. Incubate24 hrs in O2.
below.
Table 2: Brief Description of Biochemical Tests for Staphylococcus Organisms.
Motility
Catalase
Oxidase
Staphylococcus
aureus
Creamy/Tan
Medium
Facultative
Anaerobe
Non Motile
Positive
Negative
Staphylococcus
epidermidis
Creamy/Tan
Pinpoint
Facultative
Anaerobe
Non Motile
Positive
Negative
TGA
Black Colonies
Gray Colonies
Minimal Growth
Macromorphology
FTM
Coagulase
Positive
Negative
Colorless
Colorless/Pink
MSA
Colonies Yellow
Colonies Pink
Media
Media
Novobiocin
Susceptible
Susceptible
Alpha Prime or
Alpha or Alpha
Hemolysis
Beta Hemolysis Prime Hemolysis
Table 3: Probable Results for Staphylococcus Organisms.
Staphylococcus
haemolyticus
White
Small
Facultative
Anaerobe
Non Motile
Positive
Negative
Gray Colonies
Minimal
Growth
Negative
Colorless/Pink
Colonies Pink
Media
Susceptible
Alpha Prime or
Beta Hemolysis
Staphylococcus
saprophyticus
Creamy/Tan
Wavy Margin
Facultative
Anaerobe
Non Motile
Positive
Negative
Staphylococcus
xylosus
Yellow/Orange
Medium
Facultative
Anaerobe
Non Motile
Positive
Negative
Gray Colonies
Gray/Black
Colonies
Negative
Colorless
Colonies Yellow
Media
Resistant
Alpha
Hemolysis
Negative
Colorless
Colonies Yellow
Media
Resistant
Alpha
Hemolysis
Click on a link to an organism to learn more about that specific organism. Click on a link to a media test to learn
more about that specific media test. Click on a link to a specific result to see a picture and a more elaborate
description of the reaction.
2|Unknown Identification
Streptococcus
The Strep groups will do these specific tests in addition to the general tests from Table 1.
 Optochin, Bacitracin, and SXT antibiotic disks
 Hemolysis (Blood Agar)
 Hippurate hydrolysis
 Salt tolerance broth
 Bile Esculin
Test
Operating instructions
Probable Results
Optochin
Bacitracin
SXT
Use your 0.5 McFarland standard to swab half
the surface of a blood agar plate. Evenly place
one of each disk on the swabbed agar surface.
Streak the other half of the plate to check for
hemolysis. Stab into the agar surface at the last
part of your streak. Incubate for 24 hrs in CO2.
Add 5 drops of sterile water to a vial of sodium
hippurate. Add enough colonies to this
solution to give a turbid suspension. Incubate
24hrs in CO2. Add 5 drops of ninhydrin solution.
Any zone of inhibition around the Bacitracin
disk is indicative of S. pyogenes. See
probable results table 5 below.
Beta hemolysis is indicative of S. pyogenes
and S. agalactiae (sometimes). See probable
results table 5 below.
Hemolysis
Hippurate
Hydrolysis
Appearance of a purple color is indicative of
S. agalactiae and Strep faecalis. See
probable results table 5 below.
Yellow color change indicative of
Enterococcus faecalis. See probable results
table 5 below.
Blackening of the agar is indicative of S. bovis
Bile
Streak the surface of the slant. Leave the cap
and S. faecalis. See probable results table 5
Esculin
loose. Incubate for 24-48 hours in CO2.
below.
Table 4: Brief Description of Biochemical Tests for Streptococcus Organisms.
Salt
Tolerance
Lightly inoculate broth. Loosely cap and
incubate for 24-48 hours in CO2.
Streptococcus Streptococcus
agalactiae
bovis
Medium
Pinpoint
Macromorphology
Facultative
Facultative
Anaerobe
Anaerobe
FTM
Non Motile
Non Motile
Motility
Negative
Negative
Catalase
Negative
Negative
Oxidase
Resistant
Resistant
Optochin
Bacitracin
Variable
Resistant
SXT
Resistant
Variable
Gamma
Alpha
Hemolysis
Hemolysis
Hemolysis
Hippurate
Positive
Negative
Salt Tolerance
Variable
Negative
Bile Esculin
Negative
Variable
Table 5: Probable Results for Streptococcus Organisms
Streptococcus
faecalis
Medium
Facultative
Anaerobe
Non Motile
Negative
Negative
Variable
Resistant
Variable
Alpha
Hemolysis
Positive
Positive
Positive
Streptococcus
mutans
Pinpoint
Facultative
Anaerobe
Non Motile
Negative
Negative
Resistant
Resistant
Variable
Gamma
Hemolysis
Negative
Negative
Positive
Streptococcus
pyogenes
Small
Facultative
Anaerobe
Non Motile
Negative
Negative
Resistant
Susceptible
Resistant
Beta
Hemolysis
Negative
Negative
Negative
Click on a link to an organism to learn more about that specific organism. Click on a link to a media test to learn
more about that specific media test. Click on a link to a specific result to see a picture and a more elaborate
description of the reaction.
3|Unknown Identification
Gram Negative Enterics
The Enteric groups will do these specific tests in addition to the general tests from Table 1.
 Mac (MacConkey's Agar)
 MR-VP
 Urea broth
 EMB (Eosin Methylene Blue)
 Citrate
 HEA (Hekton Enteric Agar)
 TSI (Triple Sugar Iron)
Test
Operating instructions
Probable Results
Mac
EMB
HEA
Streak for isolation. Incubate 24-48 hrs at 37°C.
Streak for isolation. Incubate 24-48 hrs at 37°C.
Streak for isolation. Incubate 24-48 hrs at 37°C.
Inoculate with a single colony. Incubate 48hrs at 37°C. See media
MR-VP
tests on website for further procedures after incubation.
Citrate Streak surface only. Incubate loosely-capped 24-48hrs at 37°C.
With a needle pick the center of a well isolated colony. Stab the
center of the tube to within 3-5 mm of the bottom. Withdraw the
TSI
needle and lightly streak the surface of the slant. Incubate for 24
hrs at 37°C.
Heavily inoculate a tube of urea broth. Shake tube to distribute
Urea
organisms. Incubate for 24-48 hrs at 37°C.
Table 6: Brief Description of Biochemical Tests for Enteric Organisms.
Macro
morphology
FTM
Motility
Catalase
Oxidase
Mac
Escherichia
coli
Creamy/Tan
Medium
Facultative
Anaerobe
Motile
Positive
Negative
Pink/Purple
w/ precipitate
EMB
Black w/Green
Metallic Sheen
HEA
Yellow/Orange
w/precipitate
Poor Growth
Klebsiella
pneumoniae
Mucoid/Tan
Medium
Facultative
Anaerobe
Non Motile
Positive
Negative
Purple/Yellow
w/ precipitate
Purple maybe
Green Metallic
Sheen
Yellow/Orange
w/precipitate
Poor Growth
MR/VP
Citrate
See probable results table 7 below.
See probable results table 7 below.
See probable results table 7 below.
See probable results table 7 below.
See probable results table 7 below.
See probable results table 7 below.
See probable results table 7 below.
Proteus
vulgaris
Translucent
Diffusible
Facultative
Anaerobe
Motile
Positive
Negative
Colorless
Yellow Media
Pseudomonas
aeruginosa
Translucent
Diffusible
Motile
Positive
Positive
Colorless
Yellow Media
Salmonella
typhimurium
Creamy/Tan
Medium
Facultative
Anaerobe
Motile
Positive
Negative
Colorless
Yellow Media
Shigella
flexneri
Creamy/Tan
Medium
Facultative
Anaerobe
Non Motile
Positive
Negative
Colorless
Yellow Media
Colorless or
Pink
Colorless or
Pink
Colorless or
Pink
Colorless or
Pink
Yellow/Orange
w/Black
centers
precipitate
Clear Colonies
Blue Media
Blue/Green
w/Black
Centers
Blue Media
Clear Colonies
Blue Media
+/Positive
Red Slant
Yellow Butt
Gas, H2S
Negative
+/Negative
Unchanged
Slant
Yellow Butt
Negative
Strict Aerobe
+/+/-/+/Negative
Variable
Negative
Positive
Yellow Slant
Yellow Slant
Yellow Slant
Unchanged
Yellow Butt
Yellow Butt
Yellow Butt
TSI
Slant & Butt
Gas
Gas
Gas, H2S
Urea
Negative
Variable
Positive
Negative
Table 7: Probable Results for Gram Negative Enteric Organisms
Click on a link to an organism to learn more about that specific organism. Click on a link to a media test to
learn more about that specific media test. Click on a link to a specific result to see a picture and a more
elaborate description of the reaction.
4|Unknown Identification